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Team:Hong Kong HKU/project/at a glance
From 2013.igem.org
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Project AT a glance
Phosphate pollution in waterways and water treatment plants is a major problem. Removal of phosphate from wastewater is required to treat phosphate-containing discharge to reduce eutrophication, algal blooms and “dead zones” in lakes, rivers and coastal marine ecosystems. The aim of this project was to remove or reduce the levels of inorganic phosphate from a system or environment by employing engineered bacteria E. capsi, capable of accumulating phosphate in the form of polyphosphate. Our strategy is to express polyphosphate kinase together with the ethanolamine utilization (eut) bacterial microcompartment from Salmonella entericato provide an environment for polyphosphate synthesis. Furthermore, the project provides a novel way to recover accumulated polyphosphate, an energy rich macromolecule with many industrial uses. This paves a way towards living system-based phosphate pollution treatment to tackle critical environmental challenges.
Introduction
Bacterial MCPs are closed polyhedral shells 100-150nm diameter made of thin protein sheets, enclosing enzymes and cofactors for carbon fixation or various forms of fermentative metabolism. Recombinant Salmonella enterica ethanolamine utilization (Eut) bacterial MCP can be expressed heterologously in E.coli, both with and without the associated interior enzymes. A clonable localization N terminal signal enabling enzyme targeting to the MCP interior has been identified.
Strategy
E. capsi are genetically transformed such that they express Salmonella enterica Eut MCPs encodes by 5 structural gene EutS, M, N, L and K[2]. PPK enzyme (E.C. 2.7.4.1) from Tanneralla forsythia and Kingella oralis is cloned and fused with the signal peptide and targeted into the MCP. The engineered MCP is expected to form a sink for Pi in the system which after normal metabolic uptake by the bacteria is localized to the MCP where it is converted into polyphosphate. As the polyphosphate is too large to pass out of the pores of the MCP, it accumulates in the microcompartments, generating irreversibility in the process, and preventing phosphate return to the system/ environment. Protein modeling, based on crystallographic data, indicates that N-terminal amino acid residues of EutS shell proteins are found on the exterior surface of the MCP. To purify and recover the harvested polyphosphate, a EutS N-terminal His tag protein is genetically engineered to present exposed His tag on the exterior of the MCP. Hence the MCP containing polyphosphate can be easily purified from nickel column and the polyphosphate harvested from waste water can be utilized for multiple purposes.
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